New insights into mesoderm and endoderm development, and the nature of the onychophoran blastopore

Background Early during onychophoran development and prior to the formation of the germ band, a posterior tissue thickening forms the posterior pit. Anterior to this thickening forms a groove, the embryonic slit, that marks the anterior–posterior orientation of the developing embryo. This slit is by some authors considered the blastopore, and thus the origin of the endoderm, while others argue that the posterior pit represents the blastopore. This controversy is of evolutionary significance because if the slit represents the blastopore, then this would support the amphistomy hypothesis that suggests that a slit-like blastopore in the bilaterian ancestor evolved into protostomy and deuterostomy. Results In this paper, we summarize our current knowledge about endoderm and mesoderm development in onychophorans and provide additional data on early endoderm- and mesoderm-determining marker genes such as Blimp, Mox, and the T-box genes. Conclusion We come to the conclusion that the endoderm of onychophorans forms prior to the development of the embryonic slit, and thus that the slit is not the primary origin of the endoderm. It is thus unlikely that the embryonic slit represents the blastopore. We suggest instead that the posterior pit indeed represents the lips of the blastopore, and that the embryonic slit (and surrounding tissue) represents a morphologically superficial archenteron-like structure. We conclude further that both endoderm and mesoderm development are under control of conserved gene regulatory networks, and that many of the features found in arthropods including the model Drosophila melanogaster are likely derived. Supplementary Information The online version contains supplementary material available at 10.1186/s12983-024-00521-7.


Background
Onychophorans (velvet worms) represent a group of ecdysozoan animals that are closely related with arthropods, although it is still not fully resolved whether they represent the sister group of arthropods or the sister of arthropods + tardigrades (water bears) (recently (reviewed in Wu et al. [1]).Onychophorans, like all bilaterian animals possess three germ layers, the outer ectoderm and the two inner layers, the endoderm and the mesoderm.The outer ectoderm develops early during development from the blastula, but the inner endoand mesoderm form during gastrulation.Gastrulation is a process by which cells become internalized to form inter alia a gastric cavity that the animal uses for food digestion, and thus gastrulation is primarily the origin of the endoderm that significantly contributes to the through-gut.However, in many groups of bilaterian animals, formation of endoderm and mesoderm goes hand in hand [2].The mode of gastrulation can vary between invagination and epiboly (the ingrowing of part of the blastula cell sheet that directly leads to the formation of an outer (ectodermal) and inner (endodermal or endodermal/mesodermal) cell sheet), delamination (directional cell division of the blastula epithelium), and immigration (immigration of cells from one pole (polar immigration) or the complete surface of the blastula epithelium) (reviewed in Budd and Jensen [2][3][4]).The place of organized cell immigration (as seen in invagination, epiboly and polar immigration) is called the blastopore, a structure that is thus crucial for the formation of mesoderm and endoderm.
The origin of the onychophoran mesoderm is relatively well known [5,6].In bilaterian animals, mesoderm either forms by schizocoely or enterocoely.Enterocoely describes the direct formation (budding) of mesodermal pouches from the endodermal epithelium, the archenteron.Schizocoely describes the formation of units of mesenchymal tissue between the endoderm and the ectoderm that then each form an internal cavity, the coelom.In a subsequent step these coelomic pouches transform into an epithelial sheet.During schizocoely, typically the somites form in pairs from the posterior of the developing animal, and indeed mesoderm clearly develops by schizocoely in onychophorans: pairs of somites form on either side of the posterior pole of the embryo, the segment addition zone, that then move towards anterior underneath a layer of ectodermal cells (reviewed in Mayer et al. [6]).Together, ectoderm and somites form the early germ band that is initially split by the embryonic slit and surrounding ventral "extra-embryonic tissue" (reviewed in Treffkorn et al. [7]).
The origin of the onychophoran endoderm, however, is less well understood.Early during development, an embryonic slit develops in many groups of onychophorans that closes medially later during development [5,6,[8][9][10].The remaining anterior opening of this slit represents the onychophoran mouth and the remaining posterior opening of the slit represents the anus (thus, this structure is often referred to as the "mouth-anus furrow").Previous studies investigating morphological and genetic aspects of endoderm development revealed that definite endodermal cells are located predominantly around the embryonic slit but not the posteriorly adjacent posterior pit (the blastopore sensu Manton) (e.g.[5,8,[10][11][12][13][14]. Expression of endodermal marker genes, however, suggests that definite endodermal cells are already present anterior to the posterior pit and prior to the formation of the embryonic slit [14].Therefore, it has been suggested that both mesoderm and endoderm derive from the posterior pit and that the endoderm moves into the position around the slit, but does not originate from there [5,14]. The origin of the endoderm is important because it has a bearing on the whereabouts of the onychophoran blastopore that is generally considered the origin of endoderm development (reviewed in Technau and Scholz [2,4]).While some authors claim that the slit (or the slit plus the "posterior pit") represents the blastopore [8,11,15,16], others dispute this idea and instead suggest that only the posterior pit represents the blastopore [5,10,17,18].If the former is correct, the onychophoran mode of developmental would represent an example of an embryo that gastrulates via amphistomy (the coordinated formation of the mouth and the anus from a slit-like blastopore), and thus would support the amphistomy hypothesis (reviewed in Nielsen et al. [19]).
In this paper, we summarize our current knowledge about mesodermal and endodermal marker gene expression in the onychophoran Euperipatoides kanangrensis and provide additional gene expression data on potential endomesodermal, mesodermal and endodermal marker genes.We focus our paper on the T-box family of transcription factors that play important and conserved functions in both determination and separation of endodermal tissue from mesodermal tissue, and the differentiation of mesodermal tissues (reviewed in Showell et al. [20][21][22]).We identified three previously uninvestigated onychophoran T-box genes that all are expressed in patterns that suggest a conserved function in mesoderm and endoderm development.Beyond that, we also investigated the embryonic expression pattern of the conserved bilaterian mesoderm-and endoderm-marker genes myocyte enhancer factor-2 (mef2), Mesoderm/Mesenchyme homeobox gene (Mox), MyoD/nautilus (nau), SoxF, and B lymphocyte-induced maturation protein (Blimp) [23][24][25][26][27][28][29][30][31][32][33][34][35][36][37].Our data suggest that the gene regulatory networks that underly mesoderm and endoderm development in bilaterian animals are widely conserved also in onychophorans and that differences seen in arthropods likely represent derived features.With respect to the nature of the onychophoran blastopore, we conclude that the posterior pit indeed represents the blastoporal rim (the blastopore sensu strictu) and that the embryonic slit represents an "archenteron-like" structure.

Phylogenetic analysis
In order to detect gene orthologs in the embryonic transcriptome of the onychophoran Euperipatoides, we performed reciprocal BLAST searches (tBLASTn) using protein sequences of known orthologs from the vinegar fly Drosophila melanogaster as queries.For the detection of T-box genes we also used previously identified T-box genes from the same species as queries [10,38].Protein sequences of the detected onychophoran T-box genes were aligned with T-box genes from other metazoan species using T-Coffee (default parameters, MacVector version 12.6.0)(Nexus files: Additional files 5 and 6: Supplementary Files 5 and 6, Gene identifiers: Additional file 7: Supplementary File 7).We performed phylogenetic analyses with MrBayes [39] as previously described in [40], applying 0.75 million cycles for the Metropolis-Coupled Markov Chain Monte Carlo (MCMCMC) analysis for the main tree (Fig. 1A) and 0.3 million cycles for the tree presented in the (Additional file 1: Supplementary Fig. 1).
Mox possesses a homeodomain that is similar to that of even-skipped and Hox genes [41].Therefore, we performed a similar phylogenetic analysis as described above for T-box genes aligning the complete homeodomains of the published Hox and Even-skipped protein from Drosophila, the beetle Tribolium castaneum and Euperipatoides with the homeodomains of Tribolium Mox and Drosophila Mox (aka buttonless (btn) (Chiang et al. [42] and the putative Euperipatoides Mox protein (Additional file 8: Supplementary File 8). 1 million cycles for the MCMCMC analysis were applied.Mox proteins of Drosophila, Tribolium and Euperipatoides form a monophyletic group with almost total support that represents the sister-group of Eve proteins of these species (Fig. 1B).
We also performed a phylogenetic analysis for the zinc-finger transcription factor Blimp using confirmed orthologs of Drosophila and other species plus the best three hits found in the transcriptomes of Euperipatoides [9] and the myriapod Glomeris marginata [43], and the genomes of the common house spider Parasteatoda tepidariorum [44] and Drosophila (Additional file 9: Supplementary File 9).0.3 million cycles for the MCMCMC analysis were applied for this analysis.The here investigated Euperipatoides Blimp sequence forms a monophyletic group with confirmed Blimp proteins from other species (Fig. 1C).mef2 possesses a unique MADS box and Mef2-domain and is thus unique and unlikely to be mistaken for any other distantly related gene in the onychophoran genome [45,46].Likewise, nautilus (nau) possesses gene-specific conserved domains and thus represents another unique gene in animal genomes [23].Therefore, we did not perform phylogenetic analyses for these two genes.

PCR, gene cloning, in-situ hybridization, and nuclear staining
Total RNA from a mixed sample of embryos of different developmental stages (all stages previously defined and described in [9] was extracted using TRIZOL (Invitrogen), and reverse transcribed into cDNA using Super-Script IV RT (Invitrogen).Fragment of the identified onychophoran genes were amplified by means of RT-PCR using two sets of gene-specific primers.A nested PCRs was performed using the initial PCR as template.Primer sequences are provided in (Additional file 7: Supplementary File 7).Gene fragments of Euperipatoides Tbx15like, mef2, and nau were cloned into the PCRII vector (Invitrogen).Fragments of Euperipatoides Tbrain-like, Tbx1/org1, Blimp, and Mox were isolated with backwards primers equipped with a T7-promotor sequence overhang [47].All gene fragments were sequenced on an ABI3730XL automatic sequencer (Macrogen, Seoul, South Korea).PCR products were purified using a PCR purification kit (QIAGEN), purified PCR products of Tbrain-like, Tbx1/org1, Blimp, and Mox were used as templates for subsequent probe synthesis with T7 RNA polymerase (ROCHE).Synthesized probes were purified using the RNeasy Kit (QIAGEN).Isolation of Euperipatoides Tbx6-like, twist (twi) SoxF and H15/Tbx20 have been described previously [14,38,48].Whole mount in-situ hybridizations (WISH) were performed as described previously [48].For all genes, we investigated gene expression in embryos of stage 1-21 (staging system as introduced in Janssen and Budd [9]).All relevant expression patterns are presented in this paper.Nuclei were visualized using SYBR Green (incubation of stained embryos in 1:10, 000 SYBR Green in phosphate buffered saline with 0.1% Tween-20 (PBST-0.1%)for 20-30 min).

Thin sections
After whole mount in-situ hybridization, stained embryos of Euperipatoides were stepwise dehydrated in a series of 30% ethanol/PBS-T, 50% ethanol/PBS-T, 75% ethanol/PBS-T, 90% ethanol/PBS-T, and 99.5% ethanol/PBS-T; each step 10-15 min at room temperature (RT).Dehydrated embryos were transferred into Xylene and incubated for 10 min at RT.After this first Xyleneincubation, Xylene was exchanged and the embryos were incubated for another 10 min at RT. Xylene was removed and the embryos were transferred into melted paraffin (60 °C).Embryos were incubated in paraffin over night at 60 °C.Embryos were oriented in melted paraffin in small metal containers and transferred onto a cooling plate for approximately two hours.Embedded samples were then stored overnight (or until sectioning) in a freezer at − 20 °C.The paraffin-embedded stained embryo samples were sectioned using a Leica RM2155 Microtome.Thin sections of 6 µm were produced and transferred with a brush to the surface of a water bath (37 °C) for relaxation.Relaxed thin sections were transferred to frosted microscope glass slides.Sections were dried over night at 37 °C.After drying, the slides were deparaffinized in staining jars twice for 10 min in Xylene, and were subsequently transferred to glass jars with Clear-Rite 3 (Epredia ™ , 6901).To mount the samples, few drops of VectaMount ® Permanent Mounting Medium (Vector Laboratories, H-5000) were applied to the slides before covering them with a coverslip.The slides were left to dry on a fume bench for one to four hours.The edges of the cover slips were sealed and fixed with nail polish.

Data documentation
Photographing of stained embryos including the detection of the nuclear dye SYBR green and thin-sectioned embryos were performed under a MZ-FLIII Leica dissection microscope equipped with a Leica DC490 digital camera and an external UV-light source.Whenever indicated, linear adjustments were performed on colour, contrast and brightness using the image-processing software Adobe Photoshop 2022.The phylogenetic trees were made using FigTree V1.4.4.

Phylogenetic analysis of T-box genes
In previous studies, we published the sequences and embryonic expression patterns of four Euperipatoides T-box genes, optomotor-blind1 (omb1) (ID: c213829; HG326421), optomotor-blind2 (omb2) (ID: c205786; HG326422), Tbx20/H15 (ID: c199477; HG326423) [38], and brachyury (bra (ID: c205096, LN812023 [10]. While orthology of Tbx20/H15, bra, and omb2 was confirmed, the newly performed phylogenetic analysis revealed that the previously described omb1 gene does not represent an ortholog of omb but likely represents the ortholog of Tbx6/dorsocross (doc) (Fig. 1A).In this context, it should be stressed that this class of T-box genes usually does not form a well-supported monophyletic group in phylogenetic analyses [49][50][51][52].Likewise, in our analyses, Tbx6/doc genes, including the putative onychophoran Tbx6/doc gene, do not form a monophyletic group (Fig. 1A and Additional file 1: Supplementary Fig. 1) but fall together with Tbx4/5 orthologs, a group of T-box genes that has been secondarily lost in many animal lineages (reviewed in Sebé-Pedrós and Ruiz-Trillo [53]) (Fig. 1A).Orthology of the onychophoran Tbx6/doc gene, however is likely because all other identified T-box genes, including the newly discovered Tbx1/org1, Tbrain/Eomes, and Tbx15, fall into monophyletic groups, and thus the gene in question likely does not represent an ortholog of any of these classes of T-box genes (Fig. 1A).The previously described omb2 gene [38] thus represents the only identified omb gene in the onychophoran, therefore hereafter simply referred to as omb (Fig. 1A).
The putative onychophoran Tbx15 gene clusters with fidelity with other metazoan Tbx15 genes (Fig. 1A).While the related Tbx20/H15 and Tbx1/org1 genes are present in most metazoans, Tbx15 genes appear to have been lost in many groups of animals (reviewed in Sebé-Pedrós and Ruiz-Trillo [53]).Retention of Tbx15 has been described for the water flea Daphnia pulex and the centipede Strigamia maritima [53], their supplementary data).We tried to identify these sequences, but unique sequence identifiers were not supplied in the aforementioned publication, and they are not part of the initial study conducted by the same authors [52], their supplementary data).We were also unable to identify any arthropod Tbx15 orthologs by our own analyses including the water flea Daphnia, the spider Parasteatoda and the millipede Glomeris (Additional file 1: Supplementary Fig. 1).The discovery of a putative Tbx15 gene in the onychophoran is thus quite surprising as it appears to be the first report of a retained Tbx15 ortholog in any ecdysozoan species (note that there is neither a Tbx15 gene in the nematode worm Caenorhabditis elegans [54] nor in the priapulids Priapulus caudatus and Halicryptus spinulosus that represent a group of basally branching ecdysozoans [55] (Fig. 1A)).This onychophoran T-box gene, however, branches basally in its monophyletic group, and thus its orthology with confirmed Tbx15 genes from other species cannot be confirmed beyond doubt.It is possible that this gene represents a derived ortholog of Tbx15, or a Tbx15-like orphan T-box gene.
To our knowledge, there is no identified ortholog of Tbrain in any panarthropod species [53].One of the newly identified onychophoran T-box sequences, however, is most similar to Tbrain genes from other metazoan animals, but like for Tbx15, the onychophoran sequence is basally branching within this relatively wellsupported monophyletic group (Fig. 1A).It is therefore nevertheless possible that the gene represents a derived Tbrain ortholog or a Tbrain-like orphan rather than a true ortholog of Tbrain.
Because the phylogenetic tree of T-box genes is not resolved beyond doubt with respect to the identity of the putative onychophoran orthologs of Tbx6, Tbx15 and Tbrain, we cautiously designated these onychophoran genes as Tbx6-like, Tbx15-like, and Tbrain-like.Interestingly, however, the expression patterns of these three onychophoran genes are very much comparable with the expression patterns of confirmed Tbx6, Tbx15, and Tbrain genes in other bilaterian animals, and this indeed may be interpreted as additional support for their orthology with these genes.

Expression of onychophoran T-box genes
Tbx1/org1 is first expressed in the form of transverse segmental stripes (Fig. 2A, asterisks) and a transient diffuse pattern in the most ventral region of the developing germ band (Fig. 2A, double-arrowhead).The latter could be associated with the developing ventral nervous system.The former segmental expression is in the mesoderm (Fig. 2B).At later developmental stages, both the segmental mesodermal stripes and the diffuse ventral expression disappear from older (i.e. more anterior) segments (Fig. 2C, D).The segmental transverse stripes of expression either disappear or transform into patches of expression in the developing appendages (Fig. 2C-E).Expression in the appendages is also mesodermal, but restricted to the posterior of the appendages (Fig. 2C-E, arrows).At later developmental stages (and more anterior/older segments), expression also appears dorsal to the appendages (Fig. 2D, E, arrowheads).Within this continuous domain of expression, there is a patch of stronger expression dorsal to each appendage, except for the jaws (Fig. 2D, E, filled circles).
Tbx15-like is expressed transiently in all developing somites (Fig. 4A-F).This expression is clearly located in the mesoderm as shown by thin sections (Fig. 4E).The posterior pit, however, does not express Tbx15-like (Fig. 4A-D, dashed circle).Expression appears with the earliest onset of germ band formation and the development of the first somite (Fig. 4A).After all segments have formed, Tbx15-like remains expressed in the posterior of the embryo (Additional file 2: Supplementary Fig. 2C, D, arrowheads).
Tbrain-like is expressed prior to the formation of the embryonic slit in the center of the germ disc (Fig. 6A).Later, Tbrain-like is expressed in the embryonic slit (Fig. 6B/C, arrows), but not the posteriorly adjacent posterior pit (marked by dashed circles).When the slit closes medially, expression remains in the openings of the future mouth and anus (Fig. 6C).At this point during development, Tbrain-like is also expressed in anterior tissue of the embryo that may be associated with the developing brain (Fig. 6C, asterisks) [58] for further information on onychophoran brain development).At later developmental stages, expression in the mouth and the anus disappears, and the aforementioned anterior expression becomes either diffuse or disappears (the detected signal in (Additional file 2: Supplementary Fig. 2E) either represents weak and diffuse expression, or background; note that a similar signal is detectable in the developing appendages).
The expression of Euperipatoides twi has previously been described for early developmental stages when it is strongly expressed in the posterior pit, and weakly in the most posterior somite [14].For example at stage 4, the mesoderm of the head lobes has formed and  [9], their supplementary data).Abbreviations: hl, head lobe; j, jaw; pp, posterior pit; slime papilla weakly expresses twi (Fig. 7A, dashed circles, inlay in A, arrows), while expression in the posterior pit is much stronger (Fig. 7A, inlay in A).At later developmental stages, when the appendages begin developing, twi is expressed in the their mesoderm (Fig. 7B-I), except for the mesodermal anlagen of the nephridia (Fig. 7J) (see [59].This mesodermal expression persists in later developmental stages (Additional file 3: Supplementary Fig. 3D, E).Expression in the posterior pit is also mesodermal (Fig. 7K).Expression of twi in the frontal appendages is delayed compared to expression in the other appendages (cf.panels C and E in Fig. 7).
Expression of Mox appears in the somites at around stage 8; note that expression in the most anterior head lobe segment is somewhat delayed compared to the onset of Mox expression in more posterior segments (Fig. 8A,  B).Cross-sections reveal that this expression is clearly mesodermal (Fig. 8C).At later developmental stages, expression is seen in the entire mesoderm of the appendages, but not the overlying ectoderm (Fig. 8D-F).
Euperipatoides mef2 is strongly expressed in the ectoderm that overlays the posterior somites (Fig. 9A-D), but the posterior pit stays clear of expression (Fig. 9A-D, dashed circles).At later developmental stages, ectodermal expression becomes weak and fully disappears (Fig. 9C-F).At the same time, mesodermal expression appears in an anterior to posterior progression in the somites that later transforms into part of the limb Developmental stages are indicated (staging system after [9], their supplementary data).Abbreviations: ect, ectoderm; hl, head lobe; j, jaw; mes, mesoderm; sp, slime papilla mesoderm (Fig. 9D, F, G, arrowheads).At early developmental stages, ectodermal expression and expression in the underlying somites can co-occur (Fig. 9H).
Expression of nau starts around stage 14 in the anterior part of the mesoderm of the developing appendages (Fig. 10A-C, arrowheads).

Expression of the endodermal genes Blimp and SoxF
Before the embryonic slit forms, Blimp is already expressed in a broad domain of the blastoderm stage embryo (Fig. 11A).Later, Blimp is expressed in the embryonic slit (Fig. 11B-D, arrowheads).Soon after median closure of the slit, however, expression disappears from the future mouth region (Fig. 11D, asterisk),  [9], their supplementary data).Abbreviations: e, eye; hl, head lobe; j, jaw; L, leg; sp, slime papilla Fig. 6 Expression of Tbrain-like In all panels, anterior is to the left, ventral views.Panels A´-C´ represent SYBR Green staining of the embryos shown in panels A-C.Arrows point to expression in the embryonic slit.Dashed circles mark the position of the posterior pit.Asterisks in panel C mark expression in the the anterior tissue of the developing embryo that could be associated with the developing brain.Developmental stages are indicated (staging system after [9], their supplementary data).Abbreviations: pp, posterior pit but note that expression in the posterior region of the anus remains (Additional file 2: Supplementary Fig. 2F,  arrowhead).At stage 10, additional expression appears in the head lobes (Fig. 11D and Additional file 2: Supplementary Fig. 2F, G).At stage 13, Blimp is expressed along the ventral side of the germ band (Additional file 2: Supplementary Fig. 2F) and at later stages, this expression into segmental patches (Additional file 2: Supplementary Fig. 2G).
At the blastoderm stage, SoxF is expressed in form of a large patch anterior the forming posterior pit and before the embryonic slit forms (Fig. 12A, arrowhead).Beyond that, at this stage SoxF-expressing cells are also scattered all over the forming germ disc (Fig. 12A, arrows).Later, SoxF is expressed in the embryonic slit, but after median closure of the slit, anterior expression disappears (Fig. 12B, C, arrowheads) (also see [48].Again later during development, expression appears in the dorsal extraembryonic tissue, but unlike other markers of this tissue, SoxF is restricted to a central domain within this field of cells, suggesting that the dorsal extraembryonic tissue is not homogenous, but exhibits dorso-ventral differences (Additional file 2: Supplementary Fig. 2H).At stage 13, patches of this dorsal expression remain (Additional file 2: Supplementary Fig. 2I, asterisks) and additional expression appears in the developing nephridia (cf.[48,59] (Additional file 2: Supplementary Fig. 2I, J).

Gastrulation, and early endoderm and mesoderm induction
Ancestral functions of the T-box gene bra are tightly linked to a positive feedback loop with Wnt-signaling and likely include axis determination, suppression of neuronal genes, and endoderm specification [60,  [9], their supplementary data).Abbreviations: ect, ectoderm; fap, frontal appendage; hl, head lobe; j, jaw; L, leg; mes, mesoderm; na, nephridial anlagen; saz, segment addition zone; sp, slime papilla  Arrowheads in panels F and G point to expression in the mesoderm of developing appendages.The red bar in panels H/H´ mark the area of mesodermal expression.Developmental stages are indicated (staging system after [9], their supplementary data).Abbreviations: ect, ectoderm; hl, head lobe; L, leg; mes, mesoderm; pp, posterior pit; sp, slime papilla 61], Schwaiger et al. (2020).At least in chordates, bra is also important for the induction of the mesoderm [62][63][64][65], but it is unclear if this is a generally conserved function in other bilaterian animals (Schwaiger et al. 2020).Another likely ancestral function of bra is the regulation of genes involved in morphogenetic movements including the cells of the presumptive early mesoderm and endoderm, and thus gastrulation [4,[65][66][67].Typically, therefore bra is expressed at the place of gastrulation and the blastoporal lips, but not in the definite endo-and mesoderm.In sea urchins, for example, bra is initially expressed in endodermal precursors, but expression in these cells ceases soon after they enter the archenteron [66,68].Identified target genes of sea urchin bra are either expressed in a similar pattern as bra, or in the internalized endodermal cells that then largely do not express bra anymore [69].Similarly, during somite development in vertebrates, bra is initially expressed in the primitive streak, but is downregulated in the developing somites [70,71].In a basally branching ecdysozoan species, the priapulid worm Priapulus caudatus, expression of bra in the blastopore during gastrulation suggests a conserved function of this gene [72].Comparative data on arthropod bra (syn.brachyenteron (byn)) are surprisingly scarce outside Drosophila and other insects such as the cricket Gryllus bimaculatus and the beetle Tribolium.In all of these three insect species, byn is involved in hindgut development [73][74][75].At least in Drosophila, byn is also involved in the development of the caudal visceral mesoderm that first expresses byn strongly but stops expressing byn as the mesoderm moves anteriorly and matures (Kusch and Reuter 1999).A function of byn during gastrulation is not reported for insects.In a shrimp, however, bra expression is strongest at the gastrula stage suggesting that it may be involved in gastrulation, but whole-mount in-situ hybridization Developmental stages are indicated (staging system after [9], their supplementary data).Abbreviations: hl, head lobe data to prove that are not available [76].Since data on bry/bra expression in other arthropods are currently not available, we provide supplementary data on bra expression in the myriapod Glomeris that show that expression at the place of gastrulation and hindgut development are conserved features of bra, at least in mandibulate arthropods (Additional file 4: Supplementary Fig. 4A-D).
Another class of T-box genes that is involved in endoderm and mesoderm development is Tbx6, a target of bra reviewed i [22,77], that diverged into Tbx6, vegT and MGA in vertebrates (with vegT being lost in mammals) [53].One key function of Tbx6-class genes in vertebrates is the determination of the endomesoderm, another is organization, separation and development of endoderm and mesoderm [78][79][80][81][82][83][84][85][86], (reviewed in Wardle and Papaioannou [22]).Like bra, Tbx6-class genes are expressed in the primitive streak in vertebrates, but are not so in the forming mesodermal somites [71,87,88].Unfortunately, comparative data on Tbx6-class genes in panarthropods are restricted to the insects Drosophila and Tribolium where the Tbx6 orthologs (dorsocross (doc) genes) are not involved in gastrulation and early mesoderm and endoderm induction (reviewed in Horn and Panfilio [21,89]).
A third player in this concert appears to be Wnt11.At least in vertebrates, Wnt11 is involved in mesoderm induction, endoderm induction, convergent extension during gastrulation, axis elongation and archenteron extension [90][91][92][93][94]. Data on Wnt11 function outside the vertebrates, however, is scarce, and even expression and presence in the genome is by no means conserved in all hitherto studied species.Although Wnt11 has been lost in the lineage leading to Drosophila and the lack of early and posterior expression in Tribolium has been reported [95], at least in myriapods and some chelicerates, Wnt11 is expressed early and continuously in the posterior of the embryo, the putative site of gastrulation [96][97][98].The loss of Wnt11 in some groups of animals and the lack of Wnt11 expression during gastrulation in others may be best explained with function shuffling as reported previously for Wnt-class genes [99], Janssen et al. 2021, [100].Of interest for this study, however, is the fact that in the onychophoran Euperipatoides, Wnt11 is expressed continuously in the posterior pit in a very similar pattern as bra and Tbx6-like [101].
The expression patterns of onychophoran bra [10], Tbx6-like (Fig. 3), and Wnt11 [101] thus are comparable with their orthologs during gastrulation in most other animals: all three genes are expressed early during onychophoran development in the posterior pit, likely in endomesodermal precursors, but are not expressed in definitive mesodermal and endodermal cells as their expression is restricted to the posterior pit region.

Conserved aspects of endoderm development
We previously studied the early expression patterns of the conserved bilaterian endoderm marker genes Hepatocyte nuclear factor 4 (Hnf4), GATA456, and forkhead (fkh) in the onychophoran, and found that these genes all Fig. 12 Expression of SoxF In all panels, anterior is to the left, ventral views.Panels A´-C´ represent SYBR Green staining of the embryos shown in panels A-C.Dashed circles mark the posterior pit region.Arrows in panel A point to scattered cells that express SoxF.Arrowheads point to expression in the embryonic slit.In all panels, the posterior pit is encircled.Developmental stages are indicated (staging system after [9], their supplementary data).Abbreviations: hl, head lobe are expressed early during development in the embryonic slit suggesting that these cells represent the earliest definite endoderm [10,18].
The early expression of the newly-discovered onychophoran Tbrain-like gene is also restricted to the embryonic slit (Fig. 6).This expression appears to be comparable with the expression of Tbrain in other animals during gastrulation and endoderm development: in vertebrates, Tbrain is expressed inter alia in the early developing endoderm (reviewed in Probst and Arnold [102]), in the hemichordate Ptychodera flava, Tbrain is expressed at the base of the invaginating archenteron [103], and in the cephalochordate amphioxus and echinoderms, Tbrain is expressed in the archenteron during the process of gastrulation [104][105][106][107][108][109].Early expression and function during gastrulation is thus conserved in deuterostomes, but also in a lophotrochozoan/spiralian animal, the polychaete Hydroides elegans, Tbrain is expressed in endodermal precursors that originate from the blastoporal region [34].Therefore, Tbrain/Tbrainlike appears to represent another conserved marker of the early endoderm of bilaterian animals including onychophorans.
In bilaterian animals including chordates, echinoderms, and polychaetes, Blimp is expressed in the developing early endoderm (e.g.[28,32,110,34]).In the polychaete Hydroides elegans, for example, Blimp is expressed in the invaginated cells of the archenteron, but not the blastopore lips [34].Similarly, in the sea urchin Strongylocentrotus purpuratus and the starfish Asterina miniata, Blimp is expressed in the invaginated cells of the archenteron [32,111].In insects such as Drosophila and the cricket Gryllus bimaculatus, however, Blimp does not seem to play a role in early endoderm development [112,113].In Drosophila, expression suggests a role in the development of the precursors of the peripheral nervous system, the tracheal system, and the developing hindgut [112].Since data from other arthropods were not available prior to this study, we investigated the expression of Blimp in the myriapod Glomeris and the spider Parasteatoda.The expression of these genes suggest a conserved role of Blimp in nervous system development in arthropods, but indeed does not suggest involvement in early endoderm development (Fig. 1C and Additional file 4: Supplementary Fig. 4E-P).The observed expression pattern of Euperipatoides blimp, however, suggest that the function in nervous system development evolved in the lineage leading to Arthropoda and that the ancestral function as endoderm-patterning gene is retained in onychophorans (Fig. 11).
Expression of Euperipatoides SoxF has previously been described for mid-to-late embryonic stages when it is expressed in the posterior of the embryonic slit [48].
Because SoxF-type Sox genes are involved in endoderm development in vertebrates and are direct targets of Tbx6 [27,29,114,115], we re-investigate the expression of SoxF in the earliest accessible developmental stages of the onychophoran.In these stages, SoxF is indeed strongly expressed in the lips of the embryonic slit and the surrounding ventral extra-embryonic tissue.This suggests that SoxF may act as an early target of Tbx6 genes in vertebrates and onychophorans.If this is a result of deep conservation of the endoderm-patterning network or a result of convergence is unclear.In arthropods, however, a function of SoxF in endoderm development likely is not conserved [48,116].

Conserved aspects of mesoderm development
In onychophorans, twist (twi), a conserved regulator of mesoderm development [117][118][119][120][121][122], is expressed in the mesoderm underlying the posterior pit (Fig. 7) [14].twi is thus expressed in the earliest formed mesoderm underlying the posterior ectoderm that expresses bra and Tbx6-like.This is comparable with the situation in other animals, were bra and Tbx6/doc regulate mesodermal gene expression, but are not expressed in the definitive mesoderm.The expression of onychophoran twi, however, is in line with a conserved function in early mesoderm formation under control of bra and/or Tbx6-like.
Another potential factor of mesoderm development is Tbx15, a member of the Tbx1/15/20-class of T-box genes [123][124][125][126]. Tbx15 evolved in early metazoan lineages as it is present in sponges and ctenophores [53].In the latter, the single Tbx1/15/20 ortholog is expressed in the developing mesendodermal tissue and the edges of the blastopore [126].In the stem leading to Cnidaria + Bilateria, this gene duplicated into three separate classes, Tbx1, Tbx15 and Tbx20.While Tbx1 and Tbx20 have been retained in all lineages of Bilateria, Tbx15 has been lost from some bilaterian lineages (reviewed in Sebé-Pedrós and Ruiz-Trillo [53]).Interestingly, the onychophoran Tbx15-like gene is expressed prominently in the newly forming somites on either side of the posterior pit, and thus resembling the expression of the mesodermal marker twi, although expression of Tbx15-like in the developing somites is delayed compared to twi [14] (Figs. 4 and 13).Expression of onychophoran Tbx15-like thus is comparable with its expression in other species that retained Tbx15 such as ctenophores, cephalochordates and vertebrates [123,124,126] suggesting that the function of Tbx15 genes in early mesoderm development is conserved throughout metazoan evolution.
Mox is a conserved factor of mesoderm development and possibly also myogenesis in bilaterian animals (reviewed in Schulreich et al. [127]).In vertebrates and cephalochordates, Mox genes are transiently expressed in the forming somites early during mesoderm development [24,128], and in lophotrochozoans/spiralians, Mox is equally early expressed in the developing paired mesodermal bands Passamaneck et al. (2015), [129], Sun et al. 2022).Information on Mox expression and function in ecdysozoans is scarce.The Drosophila ortholog, buttonless (btn), is involved in mesoderm development [42], albeit at later developmental stages in the developing dorsal median (DM) cells that are of mesodermal origin [130,131].We show that this function of Mox/btn likely dates back to the last common ancestor of arthropods as revealed by the conserved pattern of Mox in the myriapod Glomeris and the spider Parasteatoda (Additional file 4: Supplementary Fig. 4Q-W).The onychophoran data thus provide the first evidence for a conserved role of Mox in early mesoderm development in any ecdysozoan species.This suggests a change of Mox function from early mesoderm development to a function in the development of the DM cells in the lineage leading Fig. 13 Schematic overview, expression of endoderm-and mesoderm-patterning genes The early expression of marker genes is shown in gene-specific colours.In the schematic drawings of embryos, anterior is pointing upwards, ventral views.Abbreviations: a, anus; dee, dorsal extraembryonic tissue; hl, somite of the head lobe segment; j, somite of the jaw-bearing segment; L, somite of a leg-bearing segment; m, mouth; m-a, mouth-anus furrow (the embryonic slit); pp, posterior pit (blastopore); saz, segment addition zone; sp, somite of the slime papilla-bearing segment; vee, ventral extraembryonic tissue to Arthropoda.The reason for this, however, remains unclear.
In Drosophila, mef2 first is expressed in all mesoderm under the control of twi, but later is restricted to certain subtypes of differentiating mesodermal tissue [45,132,133].In the crustacean Parhyale hawaiensis, twi and mef2 are not activated prior to the proliferation of segmental mesoderm, and hence comparably later than in Drosophila, the regulatory interaction of twi and mef2, however, may be conserved [134].Similarly, in spiders, mef2 appears relatively late during development in mesodermal cells including the developing heart [135,136].At least in Drosophila and Parhyale, mef2 expression is not limited to mesodermal/muscle cells, but is also expressed in ectodermal derivatives such as the developing nervous system [134,137].In Glomeris, however, mef2 is expressed early during development in the ectoderm of newly forming segments and the foregut primordium, and later also in the dorsal mesoderm and the mesoderm of the anal valves (Additional file 4: Supplementary Fig. 4X-c).The early ectodermal expression of mef2 in early forming segments of the onychophoran is thus only shared with the myriapod.The later expression in the developing and differentiating somites and the musculature of the appendages, however, likely represent conserved features of mef2 in mesoderm development (Fig. 9).
In bilaterian animals including the fly Drosophila, Tbx1 (org1) and Tbx20 (H15) genes are expressed inter alia in mesodermal derivatives such as certain types of muscles, gut parenchyme, the heart, and developing somites [21,[138][139][140][141][142][143][144].In onychophorans, Tbx1/org1 and Tbx20/H15 both are expressed in mesodermal tissue of newly formed (posterior) segments, and later also in part of the mesoderm of the developing appendages, and thus their expression is in line with a function in mesoderm development and specification as it is the case in other bilaterian animals (Figs. 2 and 5) [38].
The myogenic gene nau/MyoD generally is involved in mesoderm differentiation and muscle development in bilaterian animals [145][146][147][148].In the brachiopod Terebratalia transversa for example, MyoD first is expressed at gastrulation and early during mesoderm development, but later also persists being expressed during myogenic specification and mesoderm differentiation [149].Similarly, in the sea urchin Lytechinus variegatus, MyoD (Sum1) is expressed in mesodermal cells during gastrulation prior to myocyte differentiation [150].In Drosophila, nau is first expressed in mesodermal cells just prior to the differentiation of mesoderm into muscle precursors and the fusion of muscle cells (Michelson et al. [23].Likewise, in the myriapod Glomeris, nau is expressed relatively late during development in the prominent dorsal musculature of this animal [151].The late onset of nau expression in the onychophoran is thus in line with a conserved function of this gene in muscle differentiation. From the available published data, and the expression patterns presented in this study, we conclude that in onychophorans, twi is an early (or the earliest) marker of definitive mesodermal tissue, possibly under direct or indirect control of bra and/or Tbx6-like.The temporally staggered expression patterns of twi, Tbx15-like and Mox in the developing somites (Fig. 13) suggests consecutive functions in somite maturation and thus mesoderm differentiation.The expression of mef2, Tbx1/org1, Tbx20/H15, and nau which all are expressed in the developing mesoderm long after the onset of twi, Tbx15-like and Mox expression suggest a function in later mesoderm and muscle differentiation (summarized in Fig. 13).

The onychophoran blastopore
The conserved expression patterns reported in this paper are in line with our previous suggestions that endoderm is induced or originates from the anterior rim of the posterior pit, that mesoderm is induced or originates from the remaining part of the posterior pit, and that the posterior pit therefore represents the onychophoran blastopore [5,5,10] showed that endoderm forms already prior to the development of the embryonic slit.This corroborates with our earlier findings that endodermal markers such as Hnf4 are expressed in cells anterior to the blastopore (sensu Manton, Kennel, Janssen) before the embryonic slit forms [18].And indeed, the new data on the early endoderm marker genes Tbrain-like, SoxF and Blimp also corroborate these findings as all three genes are expressed prior to the development of a slit.It is thus very unlikely that endoderm originates from the lips of the slit, although this may indeed be the place from which a fraction of endodermal cells sink into the yolk as previously described for Euperipatoides (Eriksson and Tait [8].In addition, the persisting expression of bra, Tbx6-like and indeed also the endoderm markers forkhead (fkh) [10], Hnf4 [18] (Additional file 3: Supplementary Fig. 3A-C), GATA456 [18] and SoxF [48] anterior adjacent to the posterior pit and after closure of the embryonic slit suggests that endodermal cells still originate at later developmental stages from this region and in the absence of an embryonic slit, as previously suggested by other authors [12,13,17].Likewise, it appears that mesodermal cells still originate from the posterior pit at comparably late developmental stages as suggested by the persisting expression of the early mesodermal markers twi [14] (Additional file 3: Supplementary Fig. 3D, E) and Tbx15-like (Fig. 4).
In summary, these data suggest that the posterior pit of onychophorans represents the blastopore (blastoporal lips) and that the embryonic slit (the mouth-anus furrow) represents a unique structure of onychophoran development, likely as an adaptation to the high degree of yolk seen in most groups of onychophorans [5].Indeed, an embryonic slit does not form in yolkless placental onychophorans.Their gastrulation has been described as invagination of cells and thus formation of endoderm and mesoderm from a blastopore at the vegetal pole [17], (reviewed in Mayer et al. [6]), a process that is similar to that described for other groups of bilaterian animals.The situation in onychophorans with yolky eggs is thus likely derived.Manton speculated that "When the increase in the yolk led to the formation of a single flat disk of blastomeres lying upon the yolk, the original vegetal pole cells would lie at the periphery of the disk, …" [5].We suggest a similar but different scenario in which the posterior pit indeed represents the blastopore and that the field of cells that expresses early endodermal marker genes anterior to the posterior pit represents an archenteron-like structure that in yolkless onychophorans is internalized (as seen in many other groups of bilaterian animals), and that is located on the surface of the embryo in yolk-rich onychophorans such as Euperipatoides.
This hypothesis is testable via the investigation of spatiotemporal patterns of endodermal and mesodermal marker gene expression in the yolkless embryos of placental onychophorans.If our suggestion is true, the same endodermal markers as investigated in Euperipatoides will be expressed in internalized tissues of the gastrula in yolkless placental onychophoran embryos, and early mesodermal and endoderm/mesoderm-inducing genes will be expressed at the rim and the center of the blastopore in such embryos.

Fig. 2
Fig. 2 Expression of Tbx1/org1 In all panels, anterior is to the left.Panels A, C and D represent lateral views, dorsal up.Panel B represents a thin section.Panel E represents a dorsal view.Panel A´ represents a SYBR Green staining of the embryo shown in panel A. The double arrowheads in panels A and C point to ventral expression.The asterisks in panels A-D mark segmental expression in newly formed segments.The red asterisk in B marks the enlarged region shown in the inlay in B. The arrow in panels C and E point to expression in the limbs.Arrowheads in panels D and E point to dorsal expression.Filled circles in panel D and E mark patches of stronger expression dorsal to each appendage.Developmental stages are indicated (staging system after[9], their supplementary data).Abbreviations: ect, ectoderm; hl, head lobe; j, jaw; L, leg; mes, mesoderm; sp, slime papilla

Fig. 3
Fig. 3 Expression of Tbx6-like In all panels, anterior is to the left.Panels A and B represent ventral views.Panel C represents a lateral view, dorsal up.Panels A´-C´ represent SYBR Green staining of the embryos shown in panels A-C.The dotted line in panel A marks the embryonic slit.The arrowheads in panels A-C point to expression in the ectoderm of the posterior pit.Developmental stages are indicated (staging system after[9], their supplementary data).Abbreviations: hl, head lobe; j, jaw; pp, posterior pit; slime papilla

Fig. 4
Fig. 4 Expression of Tbx15-like In all panels, anterior is to the left.Panels A-D and F represent ventral views.Panel E represents a thin section, lateral view, dorsal up.Panels A´-D´ represent SYBR Green staining of the embryos shown in panels A-D.The dashed circles in panels A-D mark the posterior pit region.The dashed line in panels A and B mark the embryonic slit.The red asterisk in panels D and E mark the position in a whole mount (D) and a thin section E. Arrowheads and double arrowheads in panels C, D and F mark expression in the last-formed posterior somites.Developmental stages are indicated (staging system after[9], their supplementary data).Abbreviations: ect, ectoderm; hl, head lobe; j, jaw; mes, mesoderm; sp, slime papilla

Fig. 5
Fig. 5 Expression of Tbx20/H15 In all panels, anterior is to the left, lateral views, dorsal up.Panels A´ and D´ represent SYBR Green staining of the embryos shown in panels A and D. Asterisks in panels A and B mark segmental expression.Arrows in panels B-D point to mesodermal expression in the developing appendages.Panel D represents a thin section.The arrowhead in panel E marks dorsal expression in the developing heart.Developmental stages are indicated (staging system after[9], their supplementary data).Abbreviations: e, eye; hl, head lobe; j, jaw; L, leg; sp, slime papilla

Fig. 7
Fig. 7 Expression of twi In all panels, anterior is to the left, except panel J (anterior up).Panels A represents a ventral view, all other panels represent lateral views (dorsal up).Section (a) (inlay in A) shows the same embryo as in A with increased contrast to better visualize the weak most anterior expression in the somite corresponding to the head lobes (hl) (arrow).The box in panel C marks the same area as shown in panels G and H (thin sections).Panel H shows a magnification of the boxed area in panel G. Boxes in panel D show same areas as in panels I-K (thin sections).Panels A´-D´ and G´-K represent SYBR Green staining of the embryos shown in corresponding panels.Developmental stages are indicated (staging system after[9], their supplementary data).Abbreviations: ect, ectoderm; fap, frontal appendage; hl, head lobe; j, jaw; L, leg; mes, mesoderm; na, nephridial anlagen; saz, segment addition zone; sp, slime papilla

Fig. 8
Fig. 8 Expression of Mox In all panels, anterior is to the left, except panel F (dorsal view, anterior up).Panel A represents a ventral view.Panels B-E represent lateral views, dorsal up Panels A´ and C´ represent SYBR Green staining of the embryos shown in panels A and C. The dashed circle in panel A marks the region of the posterior pit.The arrow arrowhead in panel A point to expression in the last-formed posterior somites.The arrowhead in panel C (thin section) points to expression in the mesoderm.Developmental stages are indicated (staging system after[9], their supplementary data).Abbreviations: ect, ectoderm; hl, head lobe; j, jaw; L, leg; mes, mesoderm; pp, posterior pit; sp, slime papilla

Fig. 9
Fig. 9 Expression of mef2 In all panels anterior is to the left, except panel G (anterior up).Panels A, B, D and G represent ventral views.Panels C, E (thin section), F and H (thin section) represent lateral views, dorsal up.Panels A´, B´, D´, E´ and H´ represent SYBR Green staining of the embryos shown in corresponding panels.Dashed circles in panels A, B and D mark the posterior pit region.The asterisk in panel A marks enhanced expression anterior in the embryonic slit.The asterisks in panels C-E mark ectodermal expression overlying the last-formed posterior somite(s).Arrowheads in panels F and G point to expression in the mesoderm of developing appendages.The red bar in panels H/H´ mark the area of mesodermal expression.Developmental stages are indicated (staging system after[9], their supplementary data).Abbreviations: ect, ectoderm; hl, head lobe; L, leg; mes, mesoderm; pp, posterior pit; sp, slime papilla

Fig. 10 Fig. 11
Fig. 10 Expression of nau In panels A and B anterior is to the left; in panel C anterior is up.Panels A and B represent lateral views, panel C shows as ventral view.The arrowheads point to expression in the mesoderm of developing appendages.Developmental stages are indicated (staging system after[9], their supplementary data).Abbreviations: j, jaw; hl, head lobe; L, leg; sp, slime papilla